The malevolent use of biological agents as weapons by terrorist groups or rogue states continues to be a threat to the security of every country in the world. Successfully recovering bacterial bioweapons from a crime scene is an important aspect of a criminal investigation to help determine the source of the threat. In addition, the diagnosis of patients with bacterial infections in a hospital or clinical application is important.
Exploitation of pathogenic bacteria as bioweapons typically requires the release of the bacteria into the environment. Environmental releases can be accomplished using a variety of approaches. Crop duster aircraft, tank sprayers, hand sprayers, and pressurized containers can be used to disseminate aerosolized bacteria, viruses, fungi and other pathogens into the environment. Additionally, liquids or powders containing pathogens can be dispensed onto foods at restaurants, injected into foods, or used to contaminate objects handled regularly by a large number of people.
There are virtually limitless scenarios that can be envisioned for introducing pathogens into the environment. A wide array of potential surfaces can be exposed to these bioweapons. The variety of methods for disseminating bioweapons and the many surfaces and matrices that the weapons may encounter present significant challenges for effective environmental sampling for bioweapons. Nevertheless, pathogens such as bacteria remaining in the environment subsequent to a biological attack provide a potentially rich source of evidence for use in criminal investigations.
Environmental or clinical samples containing bacteria can be analyzed for target molecular signatures using polymerase chain reaction (PCR)-based methodology. PCR-based approaches often provide the first indication of the presence of bacterial threat organisms in an environmental sample. These approaches are typically highly sensitive and robust. Nevertheless, information obtained using PCR-based approaches is incomplete. The indication that any given bacterial threat agent is present in an environmental matrix is typically a consequence of obtaining positive results from several target genes from that organism.
However, using PCR-based methods alone, it is often impossible to determine if all of the targets present in the sample originate from one single bacterium, or several natural-occurring, avirulent variant organisms, each carrying a portion of the nucleic acid encoding the virulence determinants. Mechanisms of DNA exchange (including conjugation, natural transformation and DNA scavenging) and plasmid loss can occur between bacteria in nature and complicate the interpretation of molecular analyses of environmental samples. The only way to ensure that an environmental sample testing positive for a bacterial threat agent using PCR-based approaches is actually positive is to isolate and culture bacteria from the sample. Bacteria grown from environmental isolates can be rapidly identified using PCR analysis. Therefore, live culture analysis is critical for verifying the presence of target bacteria in a sample that tests positive by PCR analysis.
The successful isolation, culturing, and analysis of bacteria from environmental or patient samples provide an opportunity to gather additional types of information vital for source attribution or diagnosis purposes. Potential information obtained from successful isolation of these bacteria includes: (1) DNA sequence and genotyping data that can provide clues to the origin of the bacteria, (2) phenotypic analysis and characterization including determination of antibiotic susceptibility profiles (culture “fingerprints”), (3) serotypic characterization, (4) bacteriophage susceptibility characterization, and (5) viability/virulence of the organisms.
Therefore, bacterial weapons remaining in the environment subsequent to a biological attack provide a potentially rich source of information to support a criminal investigation. In order to be useful in this manner, however, the bacteria must be successfully isolated from the environment and cultured. Successful isolation and culturing of bacterial agents from the environment is a critical aspect of the bio forensics challenge. Therefore, it would be beneficial to have a method for culturing and growing bacteria in a manner that permitted analysis of the bacteria.
A daunting challenge of microbiology is effective culturing of fastidious bacteria from environmental sample matrices or diagnostic samples. Fastidious bacteria require specialized environments and are extremely difficult, if not impossible, to grow in a typical laboratory setting. One reason for this is the highly complex nutritional requirements of fastidious bacteria are usually impossible to replicate in the laboratory using classical culturing methods. The bacteriological threat agents of interest to governmental agencies such as the US Department of Homeland Security (DHS) have all undergone exquisite adaptation to grow as successful pathogens within the context of mammalian host systems. Isolation of these fastidious bacteria from environmental samples is difficult and the number of matrices can be quite diverse preventing a single isolation method to be employed.
In reviewing the literature relating to pathogenesis, it was found that spores of Bacillus anthracis, the causative agent of anthrax, are induced to germinate and grow robustly when placed in the environment of macrophages in vitro. B. anthracis spores placed in the culture medium used for growing macrophages (but lacking the macrophages) do not germinate. [Dixon, et. al., (2000) Cell Microbol, 2:453-463; Ireland and Hanna, (2002) Infect Immun, 70:5870-5872].
Hanna and co-workers noted that macrophages are the human body's first line of defense against infection by B. anthracis, but that during the initial stages of anthrax, the bacteria are engulfed by, and multiply within, macrophages. Those workers also reported that B. anthracis spores are induced to germinate and multiply in vitro when in the proximity of the macrophages. No engulfment, or even contact, is required for this effect, suggesting that the macrophages secrete a “factor” or “factors” to simulate multiplication of the B. anthracis in these studies. [Weiner and Hanna, (2003) Infect Immun, 71:3954-3959]. Therefore, it is evident that B. anthracis has adapted well to the host immune response; the bacteria appear to exploit the features of the immune response to enhance their own growth and disease progression.
These reports of Hanna and co-workers were observations made regarding macrophage interactions with the non-fastidious bacterium, B. anthracis, and not a process by which to isolate and grow fastidious bacteria. Fastidious bacteria including, but not limited to: Haemophilus influenza, Helicobacter pylori, Bordetella pertussis and other Bordetella species, B. fragilis, Mycobacterium tuberculosis, Legionella species, Spirochetes, Klebsiella spp., Brucella species, Francisella tularensis, Leptospira species, Borrelia burgdorferi, Bartonella species, Listeria species, Bordetella species, Eikenella corrodens, Pasteurella species, Haemophilus species, Chlamydia species, Camphylobacter species, Acinetobacter species, Mycoplasma species, Fusobacterium species, Corynebacteria species, Moraxella species, Pseudomonas species, and Neisseria species require more nuanced growth conditions.
It would be beneficial to have a method for culturing fastidious bacteria that would be applicable to fastidious pathogenic bacteria. In particular, it would be beneficial to have a method for culturing fastidious bacterial pathogens that encounter the alveolar macrophages during the initial stages of disease development. The disclosure that follows discloses one such method.